Glycoaminoglycans (GAGs) such as heparin and heparan sulfate (HS) are naturally occurring polydisperse linear polysaccharides that are heavily O- and N-sulfated (Esko et al., Annu Rev. Biochem. 2002, 71, 435-471; Gandhi et al., Chem. Biol. Drug Des. 2008, 72, 455-482). The interaction between GAGs and proteins can have profound physiological effects on hemostasis, lipid transport and adsorption, cell growth and migration and development (Capila et al., Nat. Rev. Mol. Cell Biol. 2005, 6, 530-541; Whitelock Iozzo, R. V. Chem. Rev. 2005, 105, 2745-2764; Van Vactor et al., Curr. Opin. Neurobiol. 2006, 16, 40-51; Bishop et al., Nature 2007, 446, 1030-1037; On et al., Front. Biosci. 2008, 13, 4309-4338; see also Gallagher et al., Glycobiology 1992, 2, 523-528; Spillmann et al., Curr. Opin. Struct. Biol. 1994, 4, 677-682; Rostand et al., Infect. Immun. 1997, 65, 1-8; and Sasisekharan et al., Nat. Rev. Cancer 2002, 2, 521-528. Binding of GAGs can result in the immobilization of proteins at their sites of production, regulation of enzyme activity, binding of ligands to their receptors and protection of proteins against degradation. Alteration in GAG expression has been associated with disease. For example, significant structural changes have been reported in proteoglycans surrounding the stroma of tumors and it has been suggested that these alterations may support tumor growth and invasion (Johnson et al., Cytokine Growth Factor Rev. 2005, 16, 625-636; Parish, Nat. Rev. Immunol. 2006, 6, 633-643; Taylor and Gallo, FASEB J. 2006, 20, 9-22; Chen et al., Mol. Cells 2008, 26, 415-426; Zacharski and Lee, Expert Opin. Investig. Drugs 2008, 17, 1029-1037).
Currently, more than a hundred heparan sulfate-binding proteins have been identified (Ori et al., Front. Biosci. 2008, 13, 4309-4338) and it is to be expected that in the near future many more will be discovered. For a small number of HS binding proteins, it has been established that a specific sulfation pattern is required for mediating biological activity. The best-studied case represents the interaction of antithrombin with heparin (Lindahl et al., Proc. Natl. Acad. Sci. USA 1980, 77, 6551-6555; Petitou and van Boeckel, Angew. Chem. Int. Ed. Engl. 2004, 43, 3118-3133; de Kort et al., Drug Discov. Today 2005, 10, 769-779). Each of the sulfates of the pentasaccharide GlcNAc6S-GlcA-GlcN3S-IdoA-GlcNS is essential for high affinity binding to antithrombin and anticoagulant activity. Interestingly, the pentasaccharide contains a rare glucosamine moiety that has a sulfate ester at C-3. The latter moiety is also required for binding of Herpes simplex gD protein to HS, which in turn is important for viral infection (Shukla et al., Cell 1999, 99, 13-22). On the other hand, it has been proposed that for some HS binding proteins, the spatial organization of clusters of negative charge in HS is an important determinant of binding and biological activity. It appears that in these cases, the HS binding proteins have a relaxed selectivity for short HS oligosaccharides. An example is thrombin, which requires a highly sulfated structure for binding (Grootenhuis et al., Nat. Struct. Biol. 1995, 2, 736-739). This diversity of interactions emphasizes the need for more detailed structure-activity studies on a wider range of HS binding proteins (Gama et al., Nat. Chem. Biol. 2006, 2, 467-73).
For most HS binding there is very little or no information about ligand requirements for binding and mediating biological activity (Kreuger et al, J. Cell Biol. 2006, 174, 323-327) although there is great interest in evaluating heparan sulfate variants for research and drug screening. Progress has been hampered by the difficulties of identifying HS-binding motifs for specific proteins. This difficulty is due to a lack of technology for establishing structure-activity-relations (SAR), which in turn is due to the structural complexity of natural HS and difficulties of preparing well-defined compounds (Karst and Linhardt, Curr. Med. Chem. 2003, 10, 1993-2031; Poletti and Lay, Eur. J. Org. Chem. 2003, 2999-3024; Noti and Seeberger, Chem. Biol. 2005, 12, 731-756; Linhardt et al., Semin. Thromb. Hemost. 2007, 33, 453-465; van den Bos et al., Eur. J. Org. Chem. 2007, 3963-3976; Sun et al., Prog. Chem. 2008, 20, 1136-1142). Initial approaches to establish structure-activity-relations (SAR) employed modified derivatives of heparin in which acetamido, sulfonamido, or sulfate esters were chemically modified to produce polysaccharides that have simpler compositions than the parent compound have proved useful (Yates et al., J. Med. Chem. 2004, 47, 277-280). In addition, HS has been sulfated at specific positions using biosynthetic enzymes (Lindahl et al., J. Med. Chem. 2005, 48, 349-352; Chen et al., Chem. Biol. 2007, 14, 986-993). Although these approaches make it possible to draw some conclusions about the requirement of particular functionalities for binding or biological activity, they do not allow determination of the structure of binding epitopes. Natural libraries of HS oligosaccharides have been generated and screened (Guimond and Turnbull, Curr. Biol. 1999, 9, 1343-1346) but sequencing of identified hits is still a technical challenge.
The biosynthesis of HS involves the initial formation of a simple polysaccharide composed of alternating β-D-glucuronic acid (GlcA) and α-N-acetyl-D-glucosamine (GlcNAc) units joined by 1-4 anomeric linkages. This structure is then modified by a series of enzymatic transformations involving N-deacetylation followed by N-sulfation, C-5 epimerization of GlcA to L-iduronic acid (IdoA), and finally O-sulfation. Ultimately, these modifications result in the formation of an IdoA(2-OSO3)-GlcNSO3(6-OSO3) sequence. Structural studies have, however, shown that HS contains nineteen other disaccharide sub-units arising from incomplete or additional enzymatic modifications. Combining these different disaccharides into larger structures results potentially in enormous structural diversity (Esko et al., Annu. Rev. Biochem. 2002, 71, 435-471). While it would be useful to screen a relatively large panel of well-defined HS fragments for binding to a target protein, chemical synthesis of the HS fragments has proven difficult and pain-staking. Only about 100 heparan sulfate oligosaccharides had been reported in the literature as of October, 2009, and synthesizing even a single one can take several months.
In principle, synthetic and chemoenzymatic approaches have the potential to provide a sufficiently large range of well-defined HS oligosaccharides for SAR or array development. Elegant synthetic approaches for heparin synthesis have been described (Karst and Linhardt, Curr. Med. Chem. 2003, 10, 1993-2031; Poletti and Lay, Eur. J. Org. Chem. 2003, 2999-3024; Noti and Seeberger, Chem. Biol. 2005, 12, 731-756; Linhardt et al., Semin. Thromb. Hemost. 2007, 33, 453-465; de Paz et al., Chembiochem 2001, 2, 673-685; Petitou et al., Chem. Eur. J. 2001, 7, 858-873; Codee et al., Drug Discovery Today: Technologies 2004, 1, 317-326; Lubineau et al., Chem. Eur. J. 2004, 10, 4265-4282; de Paz and Martin-Lomas, Eur. J. Org. Chem. 2005, 1849-1858; Zhou et al., Carbohydr. Res. 2006, 341, 1619-1629; Chen et al., Carbohydr. Res. 2008, 343, 2853-2862; Chen and Yu, Bioorg. Med. Chem. Lett. 2009, 19, 3875-3879; Lee et al., J. Am. Chem. Soc. 2004, 126, 476-477; Noti et al., Chem. Eur. J. 2006, 12, 8664-8686); however, no efficient strategy for the synthesis of a wide range of HS structures has been reported. Haller et al. proposed a modular approach for the chemical synthesis of a wide range of HS oligosaccharides whereby a set of properly protected disaccharide building blocks that resemble the different disaccharide motifs found in HS are assembled by a parallel combinatorial manner into larger structures (Haller and Boons, J. Chem. Soc., Perkin Trans. 1 2001, 814-822; Haller and Boons, Eur. J. Org. Chem. 2002, 2002, 2033-2038; Prabhu et al., Org. Lett. 2003, 5, 4975-4978). These efforts suffered, however, from difficulties in preparing key mono- and disaccharide intermediates, difficulties in removing temporary protecting groups, unreliability in glycosylations and difficulties in the final deprotection. Others have attempted to develop modular approaches for HS synthesis (Lee et al., J. Am. Chem. Soc. 2004, 126, 476-47; Gavard et al., Eur. J. Org. Chem. 2003, 3603-3620; Orgueira et al., Chem. Eur. J. 2003, 9, 140-169; Lu et al., Org. Lett. 2006, 8, 5995-5998; Polat and Wong, J. Am. Chem. Soc. 2007, 129, 12795-12800); however, these methods provided unnatural sulfation patterns, were unable to make structures larger than disaccharides or did not demonstrate the convenient preparation of a wide range of structural motifs. A robust strategy for the organic synthesis of a wide range of well-defined HS oligosaccharides has not been reported.